PROJECTSUMMARY/ABSTRACT Noncoding ribonucleic acid molecules called microRNAs (miRNAs) have recently emerged as important biological regulators that suppress the expression of target genes via messenger RNA degradation or translationalrepression.SincemiRNAscanregulategeneexpression,thereisintenseinterestinutilizingthese molecules as tools to halt disease progression. Unfortunately, naked miRNAs are not suitable for clinical use due to their poor stability, limited circulation half-??life, and inability to enter cells. Accordingly, researchers have begun to incorporate miRNAs into nanocarriers to facilitate their in vivo delivery. While some progress has been made, there is substantial room for improvement, evidenced by the fact that only a single miRNA nanocarrier has entered clinical trials. This lack of clinical translation indicates there is an urgent need for mechanistic studies to elucidate the underlying principles that dictate the interactions between miRNA nanocarriers and biological systems. We aim to address this need by capitalizing on our unique expertise in nanoparticle design, which includes experience with both miRNA nanocarriers and targeted nanoparticle systems.Morespecifically,wewillelucidatehowthephysicalandchemicalpropertiesofmiRNAnanocarriers influence five specific outcomes related to the challenges associated with in vivo miRNA delivery. These include: stability and nuclease resistance, cell uptake and intracellular trafficking, gene regulation potency, biodistribution, and ability to halt progression of diseases including breast cancer and osteoporosis. By studying these five outcomes, we can increase understanding of the effects of miRNA nanocarriers on the body,aswellastheeffectsofthebodyonmiRNAnanocarriers.Thiswillenableustoestablishasetofdesign rules that govern the interactions between miRNA nanocarriers and biological systems and which can be applied in the de novo synthesis of miRNA nanocarriers to maximize their site-??specific delivery and efficacy. OverthenextfiveyearswewillfocusexplicitlyonstudyinghowincorporatingtargetingagentsintomiRNA nanocarriers influences the five aforementioned outcomes. By comparing different types of targeting agents (e.g., antibodies or proteins) we can increase knowledge of the mechanisms of nanoparticle interactions with cell surface receptors and the impact they have on signal transduction. We hypothesize that targeting agents cannotonlypromotecellbinding,butalsomanipulatesignalingcascadesviareceptor-??mediatedprocesses.If this hypothesis is correct, combining miRNA delivery with targeting agent-??mediated signal cascade manipulation may have synergistic effects on diseased cells. Importantly, in the future we will expand our studiestoinvestigateotherfeaturesofmiRNAnanocarrierssuchassize,shape,andstiffness.Thiswillenable ustodistinguishhowthenanocarrieritselfinfluencesvariousbiologicaloutcomes.Thisimportantinformation willenablecreationofaccuratedesignrulesthatwillfacilitatemoreefficientclinicaltranslationofnewmiRNA nanocarriersfordiseaseintervention.